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PROGRAM BSc/Diploma in Medical Laboratory Technology SEMESTER 3 SUBJECT BLT301 - Biochemistry and Immunology - I BOOK ID B1824/B1825 SESSION Winter 2015 No Q 1 Question/Answer key Marks Total Marks 10 Name the different types of plasma proteins. Explain in detail albumin. ( Unit 6 ; Section 6.2,6.3,6.4 ) A 1 Types of plasma proteins • 1. Albumin 3 • 2. Globulin • 3. Fibrinogen Albumin • The name is derived from the term ‘albus’, which means white. A white precipitate is formed when an egg (contains the protein albumin) is boiled. It is present in high concentration in plasma, that is, about 60% of the total protein is albumin. It has one polypeptide chain with about 585 amino acids and 17 disulfide bonds having a molecular weight of 69,000. Liver produces approximately 12 grams of albumin per day. 7 • Functions of albumin • Plasma albumin performs the following functions: • 1) Osmotic function, • 2) Transport function, • 3) Nutritive function and • 4) Buffering function. Ver : BScMLT_1308 1 • Osmotic function: Albumin helps to maintain the intravascular colloidal osmotic pressure. Due to its high concentration and low molecular weight, albumin contributes to 80% of the total plasma colloidal osmotic pressure (25 mm Hg). This pressure prevents the flow of plasma into the tissue spaces by which the fluid volume inside the blood vessels is maintained. Therefore, albumin plays a major role in maintaining blood volume and body fluid distribution. The decrease in plasma albumin level leads to a fall in the osmotic pressure and in turn leads the escape of fluid into the tissue spaces resulting in ‘edema’. Edema is seen in conditions where albumin level in blood is below 2g/dl. The decrease in serum albumin is referred to as ‘hypoalbuminemia’, which is seen in the case of liver cirrhosis, nephrotic syndrome and malnutrition conditions. • Transport function: Albumin is the intravascular transporter protein for many hydrophobic substances like unconjugated bilirubin, free fatty acids, steroid hormones, thyroxine, drugs (like sulfa drugs, aspirin, salicylates, phenytoin, dicoumarol) calcium, copper and other heavy metals. • Nutritive function: Albumin serves as a source of amino acids for the tissue protein synthesis when it is broken down. In fact, albumin contains almost all proteins in good proportions. • Buffering action: All proteins show buffering action due to the presence of the amino acid histidine (Imidazole group with a pK values close to the plasma pH). Since albumin is present in high concentration in blood, and also contains good number of histidine residues, it shows maximum buffering capacity. • Hypoalbuminemia • Decrease in albumin level in blood below the normal level is termed as hypoalbuminemia. The albumin level is reduced in the blood in the following conditions: • 1.Liver diseases: Since liver synthesizes albumin, decrease in albumin level may be seen in chronic liver failure and liver cirrhosis. So, the estimation of albumin level in blood is one of the liver function tests. Since half-life of albumin is 20 days, acute liver diseases may not show decrease in albumin level. • 2.Kidney diseases: Whenever there is a defect in the kidney glomerular filtration as observed in the case of nephrotic syndrome, albumin may be excreted in urine. The condition is called ‘albuminuria’. Several gram of albumin is lost in urine daily in nephrotic syndrome. Excretion of small quantities of albumin may be seen in acute nephritis and in urinary tract infection. In normal conditions, excretion of albumin in urine is less than 150 mg/day. If more than this is excreted in urine, the condition is called microalbuminuria. Microalbuminuria is of great clinical significance since it is a predictor of future renal diseases. Albumin in urine can be detected by heat Ver : BScMLT_1308 2 and acetic acid test. In kidney diseases, edema is seen. • 3.Malnutrition/Malabsorption: In malnutrition and malabsorption syndromes, the amino acids are available in small amounts. So, there is less synthesis of albumin leading to hypoalbuminemia. There are two protein energy malnutrition disorders, namely Kwashiorkor and Marasmus. In Kwashiorkor, the serum albumin level decreases below 2g/dl, and hence edema is commonly seen. • 4.In burns and trauma, albumin may be lost through unprotected skin surfaces or through blood loss. • Albumin-Globulin ratio: The normal ratio of albumin/globulin in blood varies from 1.5:1 to 2.0:1. In hypoalbuminemia, there will be a compensatory increase in globulin level. In such cases, albumin/globulin ratio alters. In liver diseases, the ratio may be reversed. • Hypoproteinemia: Any decrease in albumin level generally decreases the total protein content of blood. Such a condition is called hypoproteinemia. So, in most of the hypoalbuminemia, conditions such as cirrhosis, nephrotic syndrome, malnutrition and malabsorption syndromes, burns and trauma, etc., hypoproteinemia is usually seen. Q 2 Define lipoproteins. Name the types of lipoproteins. Explain each lipoprotein in detail. A 2 (Unit 4; Section 4.2) 10 Definition • The plasma lipoproteins are spherical macromolecular complexes of lipids and specific proteins (apolipoproteins or apoproteins). 1 Types of lipoproteins • 1. Chylomicrons 3 • 2. Very low density lipoproteins (VLDL) • 3. Low density lipoproteins (LDL) • 4. Intermediate density lipoproteins (IDL) • 5. High density lipoproteins (HDL). Lipoproteins • Chylomicrons: These are the largest and least dense lipoproteins. They are synthesized in the intestine. They transport dietary (exogenous) triacylglycerols and cholesterol from the intestines to other tissues in the Ver : BScMLT_1308 6 3 body. They transport ingested triacylglycerols to other tissues, mainly skeletal muscle and adipose tissue, and transport ingested cholesterol to the liver. At the target tissues, the triacylglycerols are hydrolyzed by the action of lipoprotein lipase, an enzyme located on the outside of the cells that is activated by apoC-II, one of the apoproteins on the chylomicron surface. The released fatty acids and monoacylglycerols are taken up by the tissues, and either used for energy production or re-esterified to triacylglycerol for storage. As their triacylglycerol content is depleted, the chylomicrons shrink and form cholesterol-rich chylomicron remnants, which are transported in the blood to the liver. Here they bind to a specific cell-surface remnant receptor and are taken up into the liver cells by receptor-mediated endocytosis. • Very low density lipoproteins (VLDLs), intermediate density lipoproteins (IDLs) and low density lipoproteins (LDLs) are a group of related lipoproteins that transport internally produced (endogenous) triacylglycerols and cholesterol from the liver to the tissues, mainly adipose tissue and skeletal muscle. As with chylomicrons, the triacylglycerols in VLDLs are acted upon by lipoprotein lipase and the released fatty acids are taken up by the tissues. The VLDL remnants remain in the blood, first as IDLs and then as LDLs. In the transformation to LDLs, much of the cholesterol is esterified on its hydroxyl group on C-3 by the addition of a fatty acid chain from phosphatidylcholine (lecithin) by the enzyme lecithin–cholesterol acyl transferase (LCAT). In addition, all of the apoproteins other than apoB-100 are removed. • LDLs are then taken up by target cells through receptor-mediated endocytosis. The LDL receptor, a transmembrane glycoprotein on the surface of the target cells, specifically binds apoB-100 in the LDL coat. The receptors then cluster into clathrin-coated pits and are internalized. Once in the lysosomes, the LDLs are digested by lysosomal enzymes, with the cholesteryl esters being hydrolyzed by a lysosomal lipase to release the cholesterol. This is then incorporated into the cell membrane and any excess is re-esterified for storage by acyl CoA cholesterol acyltransferase (ACAT). • To prevent the build-up of cholesterol and its ester derivatives in the cell, high levels of cholesterol: • •decrease the synthesis of the LDL receptor, thereby reducing the rate of uptake of cholesterol by receptor-mediated endocytosis, and • •inhibit the cellular biosynthesis of cholesterol through inhibition of HMG CoA reductase. • High density lipoproteins (HDLs) transport endogenous cholesterol from the tissues to the liver. These have the opposite function to that of LDLs, that is, they remove cholesterol from the tissues .The HDLs are synthesized in the blood, mainly from components derived from the degradation of other lipoproteins. HDLs then acquire their cholesterol by extracting it from cell membranes and converting it into cholesteryl esters by the action of LCAT. The HDLs are then either taken up directly by the liver or transfer their Ver : BScMLT_1308 4 cholesteryl esters to VLDLs, of which about half are taken up by the liver by receptor-mediated endocytosis. The liver is the only organ that can dispose off significant quantities of cholesterol, primarily in the form of bile salts Q 3 10 Define agglutination. Mention the different types of agglutination reaction. Add a note on passive agglutination test. ( Unit 7 ; Section 7.3 ) A 3 Definition It is an antigen – antibody reaction, in which a particulate antigen combines with its antibody in the presence of electrolyte at an optimal temperature and pH, resulting in visible clumping of particles. 1 Types of agglutination reaction • 1. Slide agglutination reaction 5 • 2. Tube agglutination reaction • 3. Antiglobulin (coobs) reaction • 4. Heterophile agglutination reaction • 5. Passive agglutination reaction. Passive agglutination test • Passive agglutination test: A precipitation reaction can be converted into agglutination test by attaching soluble antigens to the surface of the carrier particles such as latex particles, bentonite and red blood cells. Such tests are called passive agglutination tests. This conversion is done because the agglutination is more sensitive for detection of antibodies. Passive agglutination tests are very sensitive. When instead of antigen, the antibody is absorbed on the carrier particles for the estimation of antigens, it is called as reversed passive agglutination. There are three methods under this test: 4 • (a)Latex agglutination test: In this polystyrene latex particles (0.8 to1µm in diameter) are widely employed to adsorb several types of antigens. Latex particles can also be coated with antibody for detection of antigen. Latex particles can also be coated with antibody for detection of antigen. These tests are very convenient, rapid and specific. These are used for detection of hepatitis B antigen and many other types of antigens. • (b)Haemagglutination test: In this test, erythrocytes sensitized with antigens are used for detection of antibodies. This test is widely used in the detection of rheumatoid arthritis and popularly known as Rose Waaler test. In rheumatoid arthritis, an auto antibody (RA factor) appears in the serum. This RA factor can agglutinate red cells coated with gamma globulins. The antigens used for the test is sheep red blood cells sensitized with rabbit antisheep erythrocyte antibody (also called as amboceptor). The amboceptor is gama globulin in nature. Ver : BScMLT_1308 5 • (c)Coagulation tests: This test is mainly based on the presence of protein A on the surface of some strains of Staphyloccus aureus (especially Cowan 1 strain). Specific IgG immunoglobulin is coated on these Cowan 1 strains of Staphylococcus aureus. ‘Fc’ portion of Ig molecule binds to protein A whereas, antigen combining ‘Fab’ terminal remains free. When the corresponding antigen is mixed with these coated cells, ‘Fab’ terminal binds to antigen resulting in agglutination. This test is most commonly used in the detection of bacterial antigens in blood, urine and CSF (Cerebrospinal Fluid).The N. gonorrhoeae, Streptococcus pyogens and H. influenza antigens can be detected by this method. Q 4 10 List the various cells of immune system. Explain lymphocytes in detail. (Unit 2; Section 2.3) A 4 Various cells of immune system • 1. Lymphocytes 3 • (a) T lymphocytes • (b) B lymphocytes • 2. Mononuclear phagocytes • 3. Granulocytic Cells • (a)Neutrophils • (b)Eosinophils • (c)Basophils • 4. Mast cells • 5. Null cells • (a)Killer cells • (b)Natural killer (NK) cell • 6. Antigen presenting cells Ver : BScMLT_1308 6 • 7.Dendritic cells Lymphocytes • Lymphocytes: 7 • They are small, non-phagocytic, mononuclear leukocytes that are immunologically competent cells.Lymphocytes are produced in the red bone marrow by the process of hematopoiesis. They leave the bone marrow, circulate in the blood and lymphatic systems, and some of them reside in the lymphoid organs. The lymphocytes are capable of recognizing a variety of foreign materials in a specific manner and consequently generate both cellular and humoral immune responses. These cells are also solely responsible for immunologic memory. Therefore, lymphocytes are considered to be the most important effector components of cellular immune system. The two major populations of lymphocytes include T and B cells. • (a)T Lymphocytes: • T cells express specific types of cell-mediated immune response carry a vast repertoire of immunologic memory, and they can regulate and directly affect a variety of immune processes. In humans, the bone marrow and thymus are the primary lymphoid organs. These are the sites of functional maturation of lymphocytes. The undifferentiated lymphocytes that migrate into the thymus undergo processing to become thymus-dependent lymphocytes or T lymphocytes. The developing lymphocytes in thymus are called thymocytes. Some T cells migrate away from the thymus and enter the blood stream where they comprise 70-80% of the circulating lymphocytes. T cells that remain in the thymus are called thymocytes. This thymus dependent differentiation of T cells occurs since early childhood and by adolescence the secondary lymphoid organs of the body generally contain a full complement of T cells. • During the maturation of T cell in the thymus, these cells express unique receptor molecule on their surface, called T-cell receptors, capable of recognizing specific antigenic structure. Based on the differences on the receptors, two well defined sub populations of T cells are evident, namely, T helper (TH) and T cytotoxic cells (TC). T cells displaying membrane-bound CD4 glycoproteins function as T helper cells, whereas those that have CD8 glycoproteins function as T cytotoxic cells. • T cells can attack (1) host cells that have been parasitized by viruses or microorganisms, (2) tissue cells that have been transplanted from one host to another, and (3) cancer cells. Since T cells must physically contact foreign or infected host cells in order to destroy them, they are said to provide cell-mediated immunity. T cells can recognize appropriate antigens only if the antigens are bound to cell membrane proteins called Major Histocompatibility Complex (MHC) molecules. There are two major types of MHC molecules viz. Class I MHC and Class II MHC molecules. Class I MHC molecules are expressed by nearly all nucleated cells, and they are composed of a heavy chain linked to a small invariant protein called 2 microglobulin. Cells that Ver : BScMLT_1308 7 display antigens complexed with a Class I MHC molecule are called altered self cells. Class II MHC molecules consist of and glycoprotein chains, and they are expressed only by Antigen Presenting Cells (APC). When a T cell encounters an antigen combined with a MHC molecule, it proliferates and differentiates into memory T cells and various types of effector T cells such as TH and TC cells. • TH cells can interact with antigens bound to Class II MHC and get activated to become effector cells, which in turn secrete various kinds of soluble immune molecules including growth factors, collectively called cytokines. The cytokines play an important role in activating B cells, T cells, macrophages and various other cell types that participate in the immune processes. Under the influence of TH derived cytokines, a TC cell that recognizes an antigen-MHC class I molecule complex proliferates and differentiates into an effector cell called Cytotoxic T lymphocyte (CTL). In contrast to TH cells the CTLs generally do not secrete many cytokines, but instead release cytotoxic molecules. • (b)B Lymphocytes: During embryonic development of mammals, B cells differentiate in the fetal liver, later in bone marrow and then released into blood circulation. The bone marrow continues to be the major site for B-cell differentiation. The letter B was originally derived from Bursa of Fabricius, an evaginated structure near the appendage of the cloaca of birds where pre-lymphocytes were first discovered to undergo functional differentiation. • B cells make up to 20-30% of the circulating lymphocytes. When the B cells leave the bone marrow they express on their membrane specific antigen recognition receptors. Such B cell receptors are antibody molecules. • Once the B cell contacts with an antigen that matches with its receptor molecule, it begins to divide rapidly and its progeny differentiates into Memory B cells and effector B cells and the latter called plasma cells. The memory B cells have a longer life span, more readily stimulated than the naive B cells, and they are retained within the body with the same receptor profile as their parent naïve B cell. Immunologic memory is the attribute of the immune system mediated by memory cells derived from B or T lymphocytes whereby a second encounter with the same antigen induces a heightened state of immune reactivity. • In contrast to memory cells derived from B-cells, the plasma cells are short lived and secrete antibodies into the blood and lymph. Antibodies are specifically directed against the antigen, which was previously responsible for B-cell stimulation leading to antibody formation. Because the antibodies are soluble immune molecules found in blood, lymph, and other extra-cellular fluids, they provide humoral immunity. The antibody-mediated humoral immune response is active mostly against the invaded bacteria, bacterial toxins and viruses. Q 5 Discuss the basis for antigen antibody reaction. Ver : BScMLT_1308 10 8 ( Unit 4 ; Section 4.2 ) A 5 Basis for antigen antibody reaction Antigen-antibody interaction is a bimolecular association similar to an enzyme-substrate interaction, with an important distinction: it does not lead to an irreversible chemical alteration in either the antibody or the antigen. Non covalent forces that are required for antigen antibody binding: (1) Ionic bond: Attraction between oppositely charged ionic groups on protein side chains can act as a binding force between the antigen and antibody. (2) Van der Waals’ force: Van der Waals’ force is generated by the interaction between the electrons in the external orbitals of two different macromolecules juxtaposed, which may be described as the attraction between the induced oscillating dipoles in the two electron clouds. (3) Hydrophobic force: When hydrophobic groups on the surface of two protein molecules come into close contact, the intervening water molecules between them are excluded. (4) Hydrogen bonding: When hydrophobic groups such as OH, NH3 and COOH are present in neighboring macromolecules, a reversible hydrogen bonding can be established between two such groups; this force is relatively weak and depends to a large extent on the closeness of the two molecules bearing these groups. Strength of Antigen Antibody Interaction: (1) Antibody Affinity: The strength of all the non- covalent interactions between a single antigen binding site on an antibody and a single epitope determines the affinity of the antibody for that epitope. (2) Antibody avidity: When complex antigens containing multiple, repeating antigenic determinants are mixed with antibodies containing multiple binding sites, the interaction of an antibody molecule with an antigen molecule at one site will increase the probability of reaction between those two molecules at a second site. Q 6 10 10 Discuss the oxidation of fatty acids and production of energy. ( Unit 2 ; Section 2.3 ) A 6 Oxidation of fatty acids β-oxidation of fatty acids: About 95% of the energy obtained from fat comes from the oxidation of fatty acids. The fatty acids in the body are mostly oxidised by β-oxidation, i.e., the oxidation on the β-carbon atom. This brings about the sequential removal of a two carbon fragment, acetyl CoA, with the production of energy in the form of ATP. The -oxidation of fatty acids takes place in three steps: 1. Activation of fatty acids occurring in the cytosol 2. Transport of fatty acids into mitochondria 3. - oxidation proper in the mitochondrial matrix. 1.Activation: Fatty acid breakdown occurs in the cytosol of prokaryotes and in the mitochondrial matrix of eukaryotes. 2. Transport into mitochondria: Activated fatty acid then gets transported into the mitochondria. 3. - oxidation proper: Oxidation: Acyl CoA undergoes oxidation by acyl CoA dehydrogenase and forms enoyl-CoA. A double bond is formed between 2nd and 3rd carbon atoms. Hydration: Enoyl-CoA hydratase brings about the hydration of double bond to form 3-hydroxyacyl CoA. Oxidation: 3-hydroxyacyl CoA undergoes second oxidation to form 3-ketoacyl Ver : BScMLT_1308 10 9 CoA producing NADH. Cleavage: Cleavage or thiolysis, of 3-ketoacyl CoA takes place in the presence of by β-ketoacyl-CoA thiolas, forming acetyl-CoA and an acyl CoA shortened by two carbon atoms. • Energy yield for each round of degradation, one FADH2, one NADH and one acetyl-CoA molecule are produced. Each NADH generates three ATP molecules, and each FADH2 generates two ATPs during oxidative phosphorylation .The yield of ATP is reduced slightly for unsaturated fatty acids, since the additional metabolic reactions, which enable them to be degraded by the β-oxidation pathway either involve using NADPH or bypass an FADH2- producing reaction. Ver : BScMLT_1308 10